The predominant mechanism in most cases of traumatic brain injury (TBI) is diffuse axonal injury (Whyte and Rosenthal, 1993). While axonal injury is common in all TBI regardless of severity (Povlishock et al., 1992; Mittl, 1994), a shearing of the axons occurs in human diffuse axonal injury (DAI) leading to progressive changes that ultimately may result in the loss of connections between nerve cells. The slow progression of events in DAI continues for up to several weeks after injury creating a window of opportunity for therapeutic intervention. Up to now, there are no consistently reproducible small animal models for DAI which closely mimic the changes associated with DAI in humans (Maxwell et al., 1997; Povlishock, 1993). Without such a model to study the mechanism of injury, it is difficult to develop prevention and/or interventional methodologies to limit the extent of injury. In part, this may explain the lack of efficacy of the clinical trials to assess various medications to limit injury in TBI.
There are approximately 500,000 new cases of TBI in the U.S. each year (Frankowski, 1985), and the incidence requiring hospitalization is estimated to be approximately 200-225/100,000 population (Frankowski, 1986; Carus, 1993). Currently, it is estimated that brain injuries account for 12% of all hospital admissions in the United States (Sandel, 1993). When compared to spinal cord injury, which accounts for less than 1% of hospital admissions, it is clear that TBI is a medical care problem which has a significant impact financially within the United States. Approximately 30,000-44,000 people will survive a severe TBI with GCS score <9 (Glasgow Coma Score Scale, Jennett, 1981) in the U.S. each year and more than 70,000 will be significantly disabled from moderate to severe TBI (GCS≦10) (Whyte & Rosenthal, 1988). Yet with new medical management techniques, less than 10% will remain in a persistent vegetative state (Whyte, 1993; Rosner, 1992; Rosner, 1990). A GCS score of eight or less generally reflects a state of unconsciousness in which the patient demonstrates no eye opening, does not follow simple commands to move muscles, and has vocalizations which are limited to sounds. Such signs are indicative of severe brain injury (Whyte, 1993; Jennett, 1975, Jennett, 1981).
Approximately 52,000 to 56,000 people die each year from TBI (Kraus et al., 1996), resulting in direct costs approximated at more than $50 billion annually (Max et al., 1991). The costs of severe TBI to the individual and family are extremely high (McMordie, 1988). Acute medical and rehabilitation bills are often around $100,000 with some considerably higher (McMordie, 1988). The Model Systems Database for Traumatic Brain Injury demonstrates there is a correlation between the average Disability Rating Score and the combined acute care and rehabilitation charges (Bullock et al., 1995). Those with a severe TBI (GCS score of 6-8) have average combined charges of $110,842, and those with a very severe TBI (GCS score 3-5) have average combined charges of $154,256 (Lehmkuhl, 1993). About one-half of all TBIs are transportation related (Whyte, 1993; Lehmkuhl, 1993) and these patients have some of the highest combined charges for acute care and rehabilitations (Lehmkuhl, 1993). This may be related to the mechanism of TBI in high speed motor vehicle crashes, specifically the presence of diffuse axonal injury (DAI) being most prevalent in the midbrain and brain stem areas (Whyte, 1993). Clearly, brain injuries of this severity that occur with high speed acceleration-deceleration injuries, have the highest costs to society. TBI clearly causes more mortality, morbidity and probably more economic loss than HIV infection in the United States.
Motor vehicle crashes of all types are responsible for approximately 40%-50% of the TBI admissions recorded in the Model TBI Systems Database (Lehmkuhl, 1993). The predominant mechanism of injury is considered to be diffuse axonal injury (DAI). Approximately 30%-40% of the fatal head injuries involve diffuse axonal injury by pathological examination (Bennett et al., 1995; McLellan, 1990). However, based on beta-amyloid precursor protein immunostaining, axonal injury may be present in all cases of fatal head injury (Gentleman et al., 1995). In cases of persistent vegetative states, Kampfl et al. (1998) recently found that all cases had evidence of DAI in magnetic resonance imaging (MRI). Diffuse axonal injury occurs even in the absence of a blow to the head and is more prevalent than previously realized. Even in mild head injury, diffuse axonal injury is present in almost ⅓ of the cases (Mittl et al., 1994). The defining characteristic of DAI is the morphologic change to the axons which occurs over the course of several days to weeks and the fact that multiple regions of the brain are injured. While a component of DAI is present in blunt or penetrating trauma injury, it is at the periphery of the injury zone and is much less significant than the predominant mechanism of injury. DAI is the major mechanism of injury in high speed acceleration-deceleration injuries associated with motor vehicle crashes. While all four mechanisms of TBI (DAI, blunt trauma, penetrating trauma, axonia) may be involved in such an injury, it is the predominant mechanism of injury under this condition.
Diffuse axonal injury is only one of the cellular mechanisms of traumatic brain injury. The others include such things as direct contusion to the cells, intracerebral hemorrhage (blood across the blood brain barrier), perfusion-reperfusion injury, and anoxia. In a high velocity TBI such as those sustained in a car accident and the subsequent sequelae one can have several mechanisms of cellular injury. Each of these mechanisms appears to cause a unique area and type of TBI. This also indicates that each type of cellular injury activates different cellular pathways and cellular channels. For instance, the sequelae of brain injury from a subtype of intracerebral hemorrhage described as subarachnoid hemorrhage (both spontaneous and traumatic) appears to respond to L-type Ca channel blockers but these same substances have not been protective in another type of TBI (European Study Group on Nimodipine in Severe Head Injury, “A Multicenter Trial of the Efficacy of Nimodipine on Outcome after Severe Head Injury”, J. Neurosurg., 1994, 80:797-804; Allen G S, Ahn, H S, Preziosi, T J, et al., “Cerebral Arterial Spasm—a Controlled Trial of Nimodipine in Patients with Subarachnoid Hemorrhage”, N. Eng. J. Med., 1983, 308:619-624). It is clear that other types of channels, including Ca channels, may be involved in other types of cellular injury.
In DAI, when enough force is applied to the cytoplasm of the neuronal cell, the elastic memory of the substance is exceeded. Then the amount of cytoplasmic deformation is directly related to the time the force is applied. This in turn relates to the amount of cytoskeletal disruption that occurs. Applicant's work has proven, with this device, that the severity of neuronal injury that occurs when a rat is injured at a defined Hertz is related to the length of time the force is applied. Furthermore, that many of the same areas of the brain have cellular disruption (corpus callosum, mesencephalon and brain stem) as is noted in humans who have suffered high velocity TBI as is noted in motor vehicle crashes. It is understood that many who have suffered a TBI in a cause similar to a motor vehicle crash may have more than one mechanism of neural cell injury. The injury inducing methods enabled by this machine will allow applicants to analyze the causes and the subsequent effects of DAI on neuronal cells and allow testing of unique compounds to protect against further neural cell death and injury without any of the other confounding, and many times masking, causes of neural cell injury being involved. In the model described the cellular disruption was not accompanied by intracerebral hemorrhage, or contusion and does not involve primarily perfusion-reperfusion or anoxic injury to the neuronal cells. By limiting the type of injury to a single type, applicant's can now study the mechanism of injury, its biochemical interactions and unique compounds to protect against neural cell injury. The foundation of the inventions in this application is its isolation of this single injury type, and the subsequently derived methods of testing for mitigation, methods of investigative research, and methods of treatment based on this uniquely isolated injury type. This is also important because DAI is, in many cases, especially in auto and other inertia caused injuries, the predominate injury type.
Many of the areas that are injured in DAI are contiguous to the areas of cerebrospinal fluid (CSF) circulation in the brain. They are thus readily accessible to treatment via diffusion with substances delivered into the CSF for circulation and such diffusion into the injured areas.
For human head injuries resulting from car collisions, the average velocity for the onset of severe injuries is 6.7 m/s (or 24.1 km/hour) as mentioned by Lorenzo et al. (1996). Most studies have been directed to the analysis of impact to the head. The Head Injury Criterion (HIC) is one method that is commonly used to assess the severity of an impact (Chou and Nyquist, 1974). Although it is considered to be the best available head injury indicator, a new finite element model using a dummy head has taken into account the effects of rotational and translational acceleration (Ueno and Melvin, 1995). Using this model, the dominant effect of translational acceleration was on principal stresses and rotational acceleration was on shear stresses.
Based on studies of head injury in primates (including man), some of the mechanical forces which bring about DAI (McLellan, 1990) have been elucidated. The crucial factors are (1) the type of acceleration/deceleration (angular rather than translational), (2) the duration of acceleration/deceleration (long rather than short), and (3) the direction of head movement (coronal rather than sagittal). Clearly angular acceleration or the associated sudden deceleration associated with an “impact” will create forces above the threshold level (McLean and Anderson, 1997). Indeed most, but not all shaken baby syndromes are characterized by a sudden deceleration (Duhaime et al., 1998).
Current research appears to point of plastic deformation within and of the axons that leads to the predominant cause of injury. The elastic tissues of the brain have plastic properties. Once the level of force is applied to a plastic substance, it is the time period over which it is applied that causes the amount of deformation. If the elastic memory of the substance is exceeded then there will be shearing and tearing. The high speed motor vehicle accident with deceleration lasting more than one to three seconds or several seconds of repetitive shaking can produce enough force for this to happen.
Materials research indicates that there is an amount of force which must be delivered below which plastic deformation of substances does not occur. In fact, the Gadd severity index initially attempted to measure the severity of injury utilizing an acceleration/time curve (Gadd, 1998). This critical amount of force appears to be essential in the development of injury (McLean & Anderson, 1997). This is very different from the contusive model of TBI where the forces are applied over milliseconds.
In nonhuman primates, this type of DAI has been induced utilizing a non-impact rotational device (Marguiles et al., 1990; Kobayashi et al., 1989). However, nonhuman primates are expensive models with significant limitations that do not lend themselves to extensive preclinical pharmaceutical and interventional trials. In rats, some DAI is found around the area of contusive injury (Meaney, 1994) but this is likely due to a small amount of localized shear forces. The sites of injury in a contusive model in rats do not conform to the areas of the brain associated with human injuries: the brain stem, corpus callosum and midbrain (Blumbergs, 1994).
More evidence is available on the mechanism of injury from the so-called “shaken baby syndrome” (Nelson et al., 1993). This mechanism of injury induces a DAI due to shaking the infant in a repeated coronal plant with or without rotational forces and there are often associated injuries to the optic nerve with this type of injury (Nelson et al., 1993). In animals, repeated coronal shaking of the head has been reported to produce some DAI utilizing miniature pigs (Ross et al., 1994; Kimura et al., 1996; Smith et al., 1997). In addition, similar histopathologic findings to the optic nerve injuries associated with the “shaken baby syndrome” have been noted after direct stretching of the optic nerves of guinea pigs (Maxwell et al., 1997).
This indicates that once the amount of force has reached a threshold, it is the length of time the force is applied with the associated plastic deformation that is the predominant factor which causes the intracellular damage to the organelles within the axon. Hence, there is a continuum over which DAI occurs in TBI. After the threshold of necessary force to create plastic deformation is reached, it may be the length of time over which it is applied that determines the amount of DAI. This would explain the findings of Foda et al. (1994) where some DAI was noted in areas adjacent to a contusion injury in rats. Unfortunately, most TBI occurs over several seconds (high speed transportation crashes) where DAI is likely to be the predominant method of injury. This is supported by the fact that many severe TBI patients have minimal changes noted on CT scan following motor vehicle crashes.
Motor vehicle crashes are the predominant cause of DAI. A component of DAI is felt to be present in all motor vehicle crashes where the patient has lost consciousness (Whyte, 1988). For many years, DAI has been known to be associated with a coma of immediate onset after brain injury, but the diagnosis could only be established by autopsy. Indeed, the clinical syndrome of coma without any preceding lucid interval, decerebration, and autonomic dysfunction were often ascribed to primary brainstem injury. However, it is now clear that primary brainstem lesions do not occur in isolation but rather in association with DAI and usually involve the cerebral hemispheres and cerebellum in addition to the brainstem (McLellan, 1990). Evidence of the mechanism of injury can be elicited by pathological studies of patients killed from high speed transportation injuries (Pounder, 1997) as well as pathological studies of “shaken baby syndrome,” a distinct subset of DAI (Nelson et al. 1993). A recent case report (Pounder, 1997) indicates that this shaking mechanism of DAI injury also applies to adults. The injury is characterized by specific neuropathological findings. On CT and MRI, this usually involves hemorrhagic punctate lesion of the corpus callosum, pontine-mesencephalic junction adjacent to the superior cerebellar peduncles and diffuse axonal damage in the white matter of the brain, brainstem and cerebellum which begin to atrophy within two weeks after injury (Whyte, 1988; Blumbergs, 1994).
Diffuse axonal injury in humans is characterized by widespread damage to axons in the cerebral hemispheres, the cerebellum and the brain stem and is a consistent feature of TBI (Adams, 1977; Adams, 1989; McLellan, 1990). The histological features of DAI depend on the length of time after injury, but within a day or so after injury there is evidence of damage to axons in the form of axonal bulbs. The initial findings are usually characterized microscopically utilizing neurofibrillar stains and stains for microglia which are abundant in the degenerating white matter. These findings are produced by the shear or flow of cytoplasm from the proximal end of a severed axon. Subsequently, the microscopic features correspond to Wallerian-type axonal degeneration as the axon disintegrates, which is probably due to metabolic disruption from injury and damage to the internal organelles from the lack of membrane integrity. In the first two years there is active myelin degeneration and in patients surviving longer, demyelination is the final stage of the process (McLellan, 1990). The result of the traumatic injury to the axons leads to the disconnection with various target sites, which is assumed to translate into the morbidity seen (Gennarelli, 1982; Povlishock, 1992). The severity of injury based on the histopathological changes has been graded in humans but not in experimental animals (Adams, 1977; Adam, 1989). The Adams classification (Adams, 1977; Adams, 1989) is used in human autopsy material, to classify the degree of DAI as mild, moderate or severe. In this classification, mild (grade 1) is characterized by microscopic changes in the white matter of the cerebral cortex, corpus callosum, and brain stem and occasionally in the cerebellum. Moderate (grade 2) is defined based on focal lesions in the corpus callosum. In severe (grade 3), there are additional focal lesions in the dorsolateral quadrants of the rostral brain stem (commonly in the superior cerebellar peduncle). This scheme has not been used for non-primate models because different regions of the brain are injured in the present models. However, it may be possible to apply this scheme to an appropriate model of DAI in small animals that is currently under development.
It has been difficult to correlate the severity of injury in humans with animal models. Animals cannot be accurately assessed by the Glasgow Coma Scale (Jennet, 1981), the Disability Rating Scale (Rappaport, 1982) or the length of post traumatic amnesia (Bishara, 1992). However, there are methods to measure the balance of animals and test their spatial memory and learning acquisition. Although non-human primates most closely resemble humans, monkeys are expensive to study. Most preclinical pharmacological studies involve rats because they are easily studied and relatively inexpensive so that large scale testing can be done. Yet, there has been no reliable reproducible rat model for DAI in the literature. There are problems, clearly the anatomy and geometry of the rat brain are less similar to the human brain than a monkey. However, by using engineering to replicate the mechanical aspects of diffuse axonal injury, the changes that occur in the rat brain are projected to be quite similar to the human condition.
The two most common animal models of human head injury are the fluid percussion and impact acceleration or weight-drop method. Fluid percussion models produce brain injury by rapidly injecting saline or blood into the closed cranium either at the midline (McIntosh et al. 1984) or laterally (McIntosh et al., 1989a). Unfortunately, these are not ideal models of human diffuse axonal injury. The models more closely replicate some of the features of subarachnoid hemorrhage. The impact acceleration (Lighthall, 1988) and the weight-drop methods (Shohami et al., 1994) both involve creating an indentation into the brain. Although some diffuse axonal injury occurs with these models, DAI is present in different areas and involve a disproportionately small volume than in humans. In order to develop a better animal model that includes diffuse axonal injury in the forebrain as is characteristic of human diffuse axonal injury, Meaney and colleagues in 1994 modified the impact-acceleration model. This new cortical impact model involves creating an indentation (1.5 mm indentation, 4.7 m/sec velocity, 22 msec dwell time) on the motor cortex combined with a contralateral craniotomy. Unfortunately, this model still lacks many features of human diffuse axonal injury. Yet another model of DAI in rats, involves dropping a weight onto a metallic disc fixed to the skull (Foda and Marmarou, 1994). Although some features of human diffuse axonal injury are seen, there are considerable amounts of brain edema and neuronal injury directly under the area of impact. This model was designed to create enough energy to reach the threshold for which some DAI will develop (McLean and Anderson, 1997). However, in models where DAI is found secondary to a contusive injury, studies directed at evaluating a treatment for DAI will be severely hindered.
In animals, repeated coronal shaking of the head has been reported to produce some DAI utilizing miniature pigs (Ross et al., 1994; Kimura et al., 1996; Smith et al., 1997). In addition, similar histopathologic findings to the optic nerve injuries associated with the “shaken baby syndrome” have been noted after direct stretching of the optic nerves of guinea pigs (Maxwell et al., 1997). In nonhuman primates, this type of DAI has been induced utilizing a non-impact rotational device (Marguiles et al. 1990; Kobayashi et al., 1989). However, nonhuman primates are expensive models with significant limitations that do not lend themselves to extensive preclinical pharmaceutical and device interventional trials.
Maxwell, Povlishock and Graham (1997) states that with the current animal models of diffuse axonal injury, axonal injury does not occur in the parasagittal white matter or corpus callosum which are the most frequent sites of axonal injury in human diffuse axonal injury. They go on to suggest that the term “diffuse axonal injury” not be used in animal models because the animal models differ from human diffuse axonal injury. The most similar model to human DAI was a primate model of DAI in which monkeys were exposed to acceleration and deceleration in the oblique, lateral and sagittal planes (Gennarelli, 1982). While the injury induced was similar to humans, primate models are prohibitively expensive when considering preclinical therapeutic interventions.
All of the clinical trials evaluating treatments for traumatic brain injury have failed. The reason for this failure may be the lack of an adequate injury model in small experimental animals such as rats and mice. An injury model that closely mimics human injury is essential in developing and evaluating treatments for patients with head injury. Applicants' efforts have been directed at producing a small animal model for the most common type of traumatic head injury called diffuse axonal injury (DAI).
The subject invention provides a small animal model that closely resembles human DAI, and essentially only DAI, in order to develop a successful treatment for DAI patients.
Similar to TBI models, in trying to develop a treatment for spinal cord injury, a critical step is to have a model that closely mimics the typical human injury. In most human spinal cord injuries, the force is directed from the anterior direction either from a burst fracture or a retropulsed disc. The force upon the spinal cord is not removed until traction or decompressive surgery can be performed. Swelling of the spinal cord occurs after injury. A new method of creating a spinal cord injury in animals has been developed to replicate the characteristics of a typical human spinal cord injury.
Heretofore, the most common model of spinal cord injury is the weight-drop method. The disadvantages of this method are: 1) the spinal cord is decompressed before injury, 2) the force is directed posteriorly, and 3) a transient force is applied upon the cord. The decompression is done before injury because it is necessary to perform a laminectomy (bone removed from the posterior surface of the spinal cord) before the weight is dropped onto the surface of the spinal cord. Removal of the bone allows room for expansion of the spinal cord as it swells after injury. The force is directed from the dorsal direction because it is very difficult to surgically approach the spinal cord from the dorsal direction especially in the case of an injury to the thoracic cord. The force upon the spinal cord is transient because the force is only from the impact of the weight. The advantage to this method is that it produces a contusive injury and that it can be replicated.
Another method that is used to injure the spinal cord is to cut the cord with a hemisection or transection. The disadvantage to this technique is that the cord in humans is only rarely cut but most often receives a contusive injury. The advantage to this method is that can easily be used to measure new growth of nerve cell processes.
Accordingly, it would be advantages and desirable to have accurate animal models of both TBI and spinal cord injury which closely approximate human TBI and spinal cord injury which overcome the drawbacks and disadvantages of the models described above and which isolates injury to essentially or substantially only DAI.